Cardioprotection by ischemic preconditioning (IP) remains an area of intense investigation. To further elucidate its molecular basis, the use of transgenic mice seems critical. Due to technical difficulty associated with performing cardiac IP in mice, we developed an in situ model for cardiac IP using a hanging-weight system for coronary artery occlusion. This technique has the major advantage of eliminating the necessity of intermittently occluding the coronary artery with a knotted suture. To systematically evaluate this model, we first demonstrated correlation of ischemia times (10–60 min) with infarct sizes [3.5 ± 1.3 to 42 ± 5.2% area at risk (AAR), Evan’s blue/triphenyltetrazolium chloride staining]. IP (4 × 5 min) and cold ischemia (27°C) reduced infarct size by 69 ± 6.7% and 84 ± 4.2%, respectively (n = 6, P < 0.01). In contrast, lower numbers of IP cycles did not alter infarct size. However, infarct sizes were distinctively different in mice from different genetic backgrounds. In addition to infarct staining, we tested cardiac troponin I (cTnI) as marker of myocardial infarction in this model. In fact, plasma levels of cTnI were significantly lower in IP-treated mice and closely correlated with infarct sizes (R2 = 0.8). To demonstrate transcriptional consequences of cardiac IP, we isolated total RNA from the AAR and showed repression of the equilibrative nucleoside transporters 1–4 by IP in this model. Taken together, this study demonstrates highly reproducible infarct sizes and cardiac protection by IP, thus minimizing the variability associated with knot-based coronary occlusion models. Further studies on cardiac IP using transgenic mice may consider this technique.
- targeted gene deletion
a cardioprotective effect by preconditioning with ischemia was first described in 1986 by Murry et al. (22), who demonstrated that pretreatment with short time periods of intermittent myocardial ischemia resulted in a marked reduction of myocardial infarct size in dogs. Since then, multiple studies have attempted to identify molecular mechanisms involved in cardioprotection by ischemic preconditioning (IP). Despite these efforts, many aspects of the molecular mechanisms involved in cardioprotection by IP remain unknown. In addition, it appears difficult to translate these concepts into a clinical setting. In fact, a profound reduction of morbidity and mortality from acute myocardial infarction, as would be expected from the initial observation (22), has not been achieved in patients yet. However, recent advances in designing transgenic mice with targeted gene deletion has revived the hope of revealing mechanisms of cardioprotection by IP. Moreover, the use of “floxed” (9, 16) or chimeric (27) mice may yield additional insight into the contribution of individual tissues or cell lines (e.g., endothelial, myeloid, or cardiac myocytes) to cardioprotection. This information may be particularly important for the design of pharmacological approaches, as pharmacokinetic requirements may differ dramatically depending on different tissues. Taken together, studies in cardiac IP using intact murine models in conjunction with targeted gene deletion may eventually unravel the molecular mechanisms responsible for cardioprotection by IP.
Despite the fact that previous studies have already successfully performed cardiac ischemia and reperfusion in mice (14, 15, 24–26), this model is technically very challenging. Particularly, visual identification of the coronary artery, placement of the suture around the vessel, and coronary occlusion by tying off the vessel with a supported knot are technically difficult. In addition, reopening the knot for intermittent reperfusion of the coronary artery during IP without causing surgical trauma adds an additional challenge. Moreover, if the knot is not tied down strongly enough, inadvertent reperfusion due to imperfect occlusion of the coronary artery may affect the results. In fact, this can easily occur due to the movement of the beating heart. On the basis of potential problems associated with using a knotted coronary occlusion system, we adopted a previously published model of chronic cardiomyopathy based on a hanging weight system for intermittent coronary artery occlusion during IP (5). In fact, coronary artery occlusion can thus be achieved without having to occlude the coronary artery by a knot. Moreover, reperfusion of the vessel can be easily achieved by supporting the hanging weights that are in a remote localization from cardiac tissues. Therefore, we hypothesized that this model would yield highly reproducible infarct sizes and cardiac protection by IP, as it combines the advantage of immediate and reliable coronary occlusion/reperfusion with avoidance of tissue trauma due to manipulation of a knot. In fact, we tested this system systematically, including variation of ischemia and reperfusion times, preconditioning regiments, body temperature, and genetic backgrounds. We found that this technique yields highly reproducible infarct sizes during murine IP and myocardial infarction. Therefore, this technique may be helpful for researchers who pursue molecular mechanisms involved in cardioprotection by IP using a genetic approach in mice with targeted gene deletion.
MATERIALS AND METHODS
Anesthesia, ventilatory support, and monitoring.
All protocols are in accordance with the German guidelines for use of live animals and were approved by the Institutional Animal Care and Use Committee of the Tübingen University Hospital and the Regierungspräsidium Tübingen. Four- to 6-wk-old C57BL/6 mice were age and gender matched. In subsets of experiments, age- and gender-matched mice with the genetic background CD1 were used. Anesthesia was induced (70 mg/kg body wt ip) and maintained (10 mg·kg−1·h−1) with pentobarbital sodium. Mice were placed on a temperature-controlled heated table (RT, Effenberg, Munich, Germany) with a rectal thermometer probe attached to thermal feedback controller to maintain body temperature at 37°C. After induction of anesthesia, mice were secured in a supine position, with the upper and lower extremities attached to the table with removable tape. The trachea was surgically exposed, and tracheal intubation was performed. In short, a blunt polyethylene cannula (Insyte 22g, Beckton Dickinson) was inserted into the trachea without direct visualization of the larynx, while the tongue of the animal was pulled out by a pair of forceps. Correct tube placement was confirmed by direct visualization of the cannula within the previously exposed trachea above the carina. The tracheal tube was connected to a mechanical ventilator (Servo 900C, Siemens, Germany) with pediatric tubing, and the animals were ventilated by using a pressure-controlled ventilation mode (peak inspiratory pressure of 10 mbar, frequency 110 breaths/min, positive end-expiratory pressure of 3 mbar, fractional inspired O2 = 0.3). Despite the fact that the Servo 900C is built as a ventilator for humans, its use in a pressure-controlled ventilator setting works excellently for the ventilation of mice. Blood gas analysis performed in studies demonstrated normal gas exchange (arterial Po2 of 115 ± 15 mmHg and arterial Pco2 of 38 ± 6 mmHg, n = 6) after 6 h of ventilation time. After induction of anesthesia, animals were monitored with an ECG (Hewlett-Packard, Böblingen, Germany). Fluid replacement was performed with normal saline, 0.1 ml/h ip. The carotid artery was catheterized for continuous recording of blood pressure with a Statham element (WK 280, WKK, Kaltbrunn, Switzerland). In brief, the carotid artery was exposed via blunt dissection of the paratracheal muscles. After further exposure and careful avoidance of any tissue trauma (particularly of the vagal nerve), a catheter was inserted into the vessel by using two sutures and a small clamp.
Technique of coronary artery occlusion.
All operations were performed under an upright dissecting microscope (Olympus SZX12). After left anterior thoracotomy, exposure of the heart and dissection of the pericardium, the left coronary artery (LCA) was visually identified. A previous study has shown that ligation of the LCA at the site of its emergence from under the left atrium results in a large myocardial infarction involving the anterolateral, posterior, and apical regions of the heart (13). For the purpose of inducing myocardial ischemia and IP, the LCA was visually identified before ligation (19). The LCA was identified by close inspection of the heart without the microscope from the side, while applying very gently pressure to the heart using a cotton stick. Once visually identified, an 8-0 nylon suture (Prolene, Ethicon, Norderstedt, Germany) was placed around the LCA. For the purpose of intermittent LCA occlusion, we adopted a chronic model of cardiomyopathy (5) by using a hanging-weight system (Fig. 1A). In short, the LCA suture was threaded through a small piece of plastic tube (PE-10 tubing) with blunt edges, and two small weights (1 g) were attached to each end. With the weights freely hanging, the LCA was immediately occluded (Fig. 1B). In addition, LCA occlusion was terminated at once, when the weights were supported. Successful LCA occlusion was confirmed by an immediate color change of the vessel from light red to dark violet (Fig. 1B), the change of color of myocardium supplied by the vessel (from bright red to white), and the presence of ST elevations in the ECG (Fig. 1C). During reperfusion, the changes of color instantly disappeared when the hanging weights were supported and the LCA was reperfused. During the whole procedure, the heart was kept wet with a wet piece of absorbent cotton.
Determination of the area at risk and myocardial infarct size.
After induction of a myocardial infarct (with or without IP), the area perfused by the LCA (area at risk, AAR) and the size of the infarct itself were determined by using a staining technique. Subsequently, infarct size was calculated as the percentage of myocardial infarction compared with the AAR. To do this, a previously described double-staining technique with Evans blue and triphenyltetrazolium chloride (TTC) was used (25). As a first step, the AAR was determined by retrograde injection of Evans blue dye into the aorta while the LCA was occluded. Thus all myocardial tissue was stained blue, except the AAR. In brief, a plastic catheter filled with heparinized normal saline was surgically inserted into the abdominal aorta. It is critical for this step to avoid air bubbles within the catheter, as they would be injected into the coronary circulation and prevent Evans blue staining. With the LCA occluded, Evans blue solution was injected retrogradely into the aorta. The heart was excised and washed in ice-cold 0.9% saline and embedded into 2% agarose. After 20 min at +4°C, the heart was cut into slices of 1 mm. The slices were incubated with 1% TTC at 37°C for 25 min. Thus the infarcted area is demarcated as a white area, while viable tissue stains red. The stained slices were fixed with 10% formaldehyde overnight. The AAR and the infarct size were determined via planimetry by using the NIH software Image 1.0 (8), and the degree of myocardial damage was calculated as the percentage of infarcted myocardium from the AAR.
As a first step, the influence of different ischemia times (10, 20, 30, 45, and 60 min) on infarct size were investigated. As it is not entirely clear from previous studies (8) how much reperfusion time is necessary in mice for successful TTC staining, different reperfusion times (30, 60, 90, 120, and 240 min) were used after a constant ischemic period. Next, the influence of different genetic backgrounds (C57BL/6 and CD1) and body temperatures (37°C and 27°C) were examined. For cold ischemia, mice were kept at 27°C and rewarmed to 37°C during reperfusion. Cardioprotection by IP was demonstrated in this model, and different IP regimens with different numbers of ischemia-reperfusion cycles were tested.
Cardiac enzyme measurement.
As an additional readout for myocardial infarct severity, we measured cardiac troponin I (cTnI) levels in the plasma of mice with or without IP. In short, blood was obtained from the portal vein following indicated treatment (myocardial ischemia of 60 min with or without prior IP, 4 × 5 min ischemia, followed by 5 min of reperfusion). Plasma cTnI levels were determined with a quantitative rapid cTnI assay (Life Diagnostics, West Chester, PA).
Gene regulation by cardiac IP.
To test the usefulness of this model to assess transcriptional consequences of IP, we used real-time RT-PCR in this model to demonstrate regulation of a group of genes known to be hypoxia regulated. In fact, we had shown in previous work that equilibrative nucleoside transporter (ENT) 1 and ENT2 are repressed by hypoxia (7). Under the assumption that IP would also repress these genes, we performed four cycles of IP (5 min ischemia, 5 min reperfusion) and excised the AAR after indicated time periods followed by isolation of RNA, reverse transcription, and quantification with ENT-specific primers by real-time RT-PCR (iCycler; Bio-Rad Laboratories, Munich, Germany). Additionally, the gene regulation of the other equilibrative nucleoside transporters (ENT3 and ENT4) was assessed. In short, total RNA was isolated from heart tissue using the total RNA isolation NucleoSpin RNA II Kit according to the manufacturer’s instructions (Macherey and Nagel, Düren, Germany). For this purpose, tissue from the AAR was homogenized in the presence of RA1 lysis buffer (Micra D8 homogenizer, ART-Labortechnik, Müllheim, Germany), and after filtration, lysates were loaded on NucleoSpin RNA II columns, followed by desalting and DNase I digestion (Macherey and Nagel). RNA was washed, and the concentration was quantified. cDNA synthesis was performed by using reverse transcription according to the manufacturer’s instructions (i-script Kit, Bio-Rad Laboratories). The primer sets for the PCR reaction contained 10 pM sense and 10 pM antisense with SYBR Green I (Molecular Probes). Primer sequences for murine ENT1, ENT2, ENT3, and ENT4 were the sense and anti-sense primers 5′-CTTGGGATTCAGGGTCAGAA-3′ and 5′-ATCAGGTCACACGACACCAA-3′, 5′-CATGGAAACTGAGGGGAAGA-3′ and 5′-GTTCCAAAGGCCTCACAGAG-3′, 5′AACCTGGGCTACAGGAGACA-3′ and 5′-TAGAACAGGGAGCCCTGAGA-3′, and 5′-AGGGGGCGTTTATTCAGTCT-3′ and 5′-AGAACGGAGTTGGGGACTTT-3′, respectively. The primer set was amplified by using increasing numbers of cycles of 94°C for 1 min, 58°C for 0.5 min, and 72°C for 1 min. Murine β-actin (sense primer, 5′-ACATTGGCATGGCTTTGTTT-3′ and antisense primer, 5′-GTTTGCTCCAACCAACTGCT-3′) in identical reactions were used to control for the starting template.
Data were compared by two-factor ANOVA or by Student’s t-test where appropriate. Values are expressed as means ± SD from 4–6 animals per condition.
Influence of ischemia time on myocardial infarct size.
Because of the fact that previous studies in murine cardiac IP suggest different ischemia times (14, 17, 23), we first tested the effect of different ischemia times on infarct size in this model. In fact, for studies of cardioprotective effects of IP, it would be ideal to use an ischemia time associated with an in infarct sizes of approximately 30 to 40% of the AAR. Thus it is would be possible to demonstrate changes in both directions, e.g., smaller infarct sizes with cardiac IP or larger infarct sizes with an experimental therapeutic or a specific gene deletion. In addition, mice with an infarct size of <50% usually survive the experiment, whereas infarct sizes of 60–80% are often not survived and the animals die before the reperfusion time is complete. As shown in Fig. 2, 10 min of myocardial ischemia followed by 2 h of reperfusion resulted in an infarct size of 3.5 ± 1.3% of the AAR. In contrast, an ischemia time of 60 min resulted in a mean infarct size of 42 ± 5.2% of the AAR (P < 0.01). Over the examined range (10–60 min), ischemia time correlated with infarct size (R2 = 0.97, P < 0.001). Taken together, these results demonstrate that the use of the hanging-weight system for murine coronary ischemia is associated with highly reproducible infarct sizes closely correlating with ischemia time.
Influence of reperfusion time on the documentation of infarct size by TTC staining.
After having shown that the time of coronary artery occlusion closely correlates with myocardial infarct size, the next step was determination of the required reperfusion time for TTC staining in this model. In fact, the reperfusion time is critically important for TTC staining. The colorless dye is reduced to a brick red-colored precipitate by dehydrogenases in the presence of the coenzyme NADH. Dying cells lose their ability to retain NADH and, therefore, are delineated as pale areas within the red-stained viable myocardium. Infarct size delineation by TTC requires that NADH has been washed out completely from the necrotic area. However, if reperfusion is not long enough, infarct size delineation by TTC staining may result in an underestimation of the actual infarct size (11). Therefore, we investigated the required reperfusion time for correct infarct size delineation by TTC staining in this model. Using an ischemia time of 60 min, we tested reperfusion times of 30–240 min. As shown in Fig. 3, despite similar ischemia times (60 min), the infarct size measurement increased from 11.5 ± 4.5% after 30 min to 42. ± 5.1% after 120 min. No further increase in infarct size could be detected with longer reperfusion times (240 min). These results highlight that at least 2 h of reperfusion time in this model of murine myocardial ischemia is necessary for reliable infarct staining by TTC. On the basis of these results, all further studies were performed with 2 h of reperfusion time.
Effect of genetic background.
On the basis of previous reports suggesting that the size of myocardial infarction after a defined ischemic period may differ between different backgrounds in rats (1), we compared infarct sizes in two different genetic murine backgrounds with the hanging-weight system of coronary occlusion (C57BL/6 and CD1). For this purpose, 60 min of ischemia time followed by 2 h of reperfusion were applied. Infarct size was measured by Evans blue/TTC double staining. As shown in Fig. 4A, C57BL/6 mice had an infarct size of 45.0 ± 6.1% of the AAR compared with 66.8 ± 6.7% in CD1 mice (P < 0.01). Taken together, these data demonstrate marked differences between different genetic backgrounds in murine. In fact, these underline the critical importance of performing control experiments in closely matched littermate controls of a similar genetic background.
Effect of body temperature.
In additional experiments, we assessed the effect of myocardial ischemia on infarct size at different body temperatures in this model. We usually maintain the rectal temperature of animals at 37°C by using a heated operating table. With these settings, 60 min of murine myocardial ischemia followed by 2 h of reperfusion were associated with an infarct size of 43 ± 5.2% of the AAR. In contrast, animals that were maintained at a body temperature of 27°C until the end of ischemia and then rewarmed to 37°C had an infarct size of 7.2 ± 4.6% AAR (Fig. 4B, *P < 0.01). These results demonstrate cardioprotection during myocardial ischemia by hypothermia and highlight the critical role of maintaining the same body temperature between experimental groups.
Influence of IP on infarct size.
After having demonstrated reproducible infarct sizes with myocardial ischemia and reperfusion time, we next used the hanging-weight coronary occlusion system in experiments of cardioprotection by IP. We first used four cycles of IP (5 min ischemia, 5 min reperfusion, Fig. 5A), followed by an ischemia time of 60 min and a reperfusion time of 2 h. Under these conditions, IP was associated with a 3.2-fold reduction of infarct size from 42.2 ± 5.1% to 13.3 ± 3.3% of the AAR (P < 0.01, Fig. 5, B and C). To test the influence of different cycle numbers, we performed IP with 5 min of ischemia and 5 min of reperfusion at increasing cycle numbers. As shown in Fig. 6, cardioprotective effects correlate with the number of cycles (R2 = 0.81, P < 0.05). No significant infarct size reduction was seen with one or two numbers of cycles. Only after three or four cycles of IP was a significant infarct size reduction detected (P < 0.01). These results demonstrate cardioprotection by IP in this model. Consistent with previous work, we confirm here that at least two cycles of IP are required for cardioprotection (2).
Influence of cardiac IP on plasma troponin I levels.
In all previous experiments, infarct severity was assessed by measuring infarct sizes via TTC staining and expressing them as percentage of the AAR. To obtain an additional readout, we used cTnI levels. For this purpose, we performed 60 min of ischemia and 2 h of reperfusion with or without IP. As shown in Fig. 7A, the cTnI plasma levels were significantly lower in the IP-treated group (P < 0.01). Consistent with previous studies on cardiac troponin (18), infarct sizes correlated closely with cTnI plasma levels (r2 = 0.8, P < 0.05). These studies demonstrate that in addition to measuring infarct sizes after TTC/Evans blue staining, measurement of myocardium-specific enzymes (cTnI) give a reliable readout for infarct severity in this model.
Modulation of gene expression by cardiac IP.
As the last step, we measured gene regulation by IP in this model. For this purpose, we performed IP as shown in Fig. 8A and excised the AAR after 90 min. ENTs are highly expressed in the cardiovascular system, but very little is known about their role in cardiomyocyte physiology (3, 4). In addition, previous studies also suggest that dipyridamole, an inhibitor of ENT1 (and to a lesser extent ENT2) significantly potentates the infarct size-limiting effect of preconditioning (21). Similar to what is known about ENT1 and ENT2 regulation by hypoxia (7), we could show here that ENT1 and ENT2 are transcriptionally repressed 90 min after IP (*P < 0.01 compared with control; Fig. 8B). This observation suggests that ENT repression by IP may serve as a mechanism of cardioprotection by IP. Although only little is known about the role of ENT3 and ENT4 in cardiac disease, we show here that they are also transcriptionally repressed by IP. Taken together, these results highlight the usefulness of this model to measure transcriptional effects of cardiac IP.
In all experiments, heart rates and blood pressure were monitored. Except for the mice with cold ischemia (mean heart rate of 180 ± 60 beats/min), the heart rates were constant with a mean value of 480 ± 60 beats/min. Mean blood pressures levels were always higher than 60 mmHg throughout the experiment. No differences in blood pressure were observed between mice treated with IP and control animals. Blood pressure was significantly reduced after ischemia in both groups (P < 0.01), most likely reflecting decreased cardiac output after the acute myocardial infarction (Table 1).
The investigation of cardioprotection by IP is currently an area of intense investigation. To further advance research in this area, the use of genetically engineered animals, particularly of transgenic mice, may provide additional insight into molecular mechanisms of cardioprotection by IP. In fact, this approach may yield more convincing and more detailed information regarding the contribution of specific gene products to cardioprotection than previous pharmacological studies. Because of the technical difficulty associated with manually interrupting the blood flow of a murine coronary artery by tying a supported knot, we adopted a knot-free hanging-weight system for coronary occlusion. In the present study, we systematically tested this model using different protocols, including different ischemia-reperfusion times and different genetic backgrounds. In addition, we tested different cardioprotective regiments, including hypothermia and different IP protocols. Finally, we also demonstrated regulation of a specific gene group by preconditioning in this model. Taken together, these data provide feasibility of this model, thus minimizing the variability associated with knot-based coronary occlusion models. Therefore, this technique may be very useful for the study of cardioprotection by IP in murine models.
Similar to the present study, previous investigations have shown that cardioprotective effects of IP can be demonstrated in mice (8, 10, 20, 26). In fact, similar to larger animals, mice show a robust infarct-sparing effect during both an early and a late phase of cardiac IP, although the early phase is more powerful (10). In addition, some studies were already able to demonstrate absence of cardiac IP in mice with targeted deletion of a specific gene (15, 25). For example, Schwanke et al. (25) were able to demonstrate that heterozygote mice with targeted deletion of the gap-junction protein connexin 43 do not show decreased infarct sizes on IP. These studies were performed by using a knot system to occlude the coronary artery. Despite these technically successful studies of murine cardiac ischemia and reperfusion, we believe that using a knot-free system of coronary artery occlusion may be superior and yield more reliable and reproducible results. In fact, it has been our experience with knotted systems in mice that it is hard to guarantee reliable coronary occlusion during ischemia. In fact, the movement of the beating heart can cause inadvertent opening of the knot, resulting in undetected coronary reperfusion. Alternatively, when tying down the knot so tight that coronary occlusion is secured, reopening of the knot during intermittent reperfusion as required for IP without tissue trauma may be difficult.
Despite many advantages associated with using targeted gene deletion in mice for studying cardioprotection, some limitations of this approach have to be pointed out. Although it is likely that the use of genetically modified mice may yield important information about IP, biological compensation for gene deletion is known to occur. Moreover, it is well appreciated that different responses to myocardial ischemia have been observed, not only with regard to different genetic backgrounds, but also between different species (6). In fact, a recent study compared closed-chest models of canine and mouse infarction/reperfusion qualitatively and quantitatively. Much like the canine model, reperfused mouse hearts showed a marked induction of endothelial adhesion molecules, cytokines, and chemokines. In contrast, reperfused mouse infarcts showed accelerated replacement of cardiomyocytes by granulation tissue, leading to a thin mature scar at 14 days, when the canine infarction was still cellular and evolving. In addition, infarcted mouse hearts demonstrated a robust postreperfusion inflammatory reaction, associated with upregulation of different cytokines. Taken together, the postinfarction inflammatory response and resultant repair in the mouse heart share many common characteristics with large mammalian species but has distinct temporal and qualitative features (6). Such studies underscore that despite multiple similarities, molecular mechanisms and potential therapeutic targets identified in murine models cannot always be directly transferred into a clinical scenario but may first require further testing in other models.
An additional limitation of using a murine model of cardiac IP is the technical difficulties associated with this model. In our experience, it takes at least 6 mo of continuous surgical training with this model before reproducible infarct sizes and consistent cardioprotective effects by IP can be achieved. In addition, it is practically impossible to sample blood from distinct anatomic locations, e.g., from the coronary sinus or pulmonary artery, or to modulate pharmacological conditions in a more specific fashion (e.g., intracoronary application). Therefore, it is extremely difficult to obtain detailed physiological data in this model. An alternate approach that allows better control of the specific environment and physiological conditions is Langendorff preparations. For example, myocardial preload and afterload can be easily modulated in this model, and physiological parameters such as the left ventricular developed pressure or the rate-pressure product can be obtained and put into context of IP (12). Nevertheless, it remains unclear how well an excised heart inserted into a Langendorff apparatus truly reflects the physiological conditions of myocardial ischemia and reperfusion.
In summary, the present study describes a novel technique of performing IP in an intact murine model by using a hanging-weight system and thus avoiding coronary artery occlusion by a knot. In fact, this study demonstrates highly reproducible infarct sizes and cardiac protection by IP, thus minimizing the variability associated with knot-based coronary occlusion models. Investigators who consider studying cardioprotection by IP in mice may benefit from this model.
This work was supported by Fortune Grant 1416–0-0 and Interdisciplinary Center for Clinical Research (IZKF) Verbundprojekt 1597–0-0 from the University of Tübingen, Germany and German Research Foundation (DFG) grant EL274/2–2 to H. K. Eltzschig, IZKF Nachwuchsgruppe 1605–0-0 of the University of Tübingen, Germany to T. Eckle, and Grant 01 KS 9603 from the Ministry for Education and Research of the Federal Republic of Germany, IZKF, Würzburg, Germany to A. Redel.
We acknowledge Stephanie Zug and Marion Faigle for technical assistance, and Shelley K. Eltzschig for artwork during manuscript preparation.
↵* T. Eckle and A. Grenz contributed equally to this study.
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- Copyright © 2006 by the American Physiological Society